Truncated Cas9 Protein for AAV Packaging in Viral Genome Editing
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Solution Overview
Problem
Current gene therapy approaches using zinc finger nucleases (ZFNs) and CRISPR/Cas9 systems face limitations such as unwieldiness, low specificity, and the inability to target multiple DNA sites simultaneously, particularly due to packaging constraints of adeno-associated virus (AAV) vectors, which restricts their application in treating viral infections and cancers.
Innovation Solution
Development of smaller Cas9 proteins, like Staphylococcus aureus Cas9, and the use of recombinant constructs with single guide RNAs (sgRNAs) to target multiple DNA sequences, enabling efficient gene editing and deletion, including viral genome elimination, through CRISPR/Cas9 systems delivered via AAV vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Streptococcus pyogenes Cas9 RGN is used for targeted DNA cleavage, then high specificity and multiplex targeting capability are achieved, but the protein size exceeds the packaging limit of AAV vectors
Solution Approach 1:
The patent extracts and removes specific amino acid residues from the S. pyogenes Cas9 protein structure to create a truncated version. Specifically, residues 1-10 and 2048-2370 are removed, reducing the molecular weight from approximately 160 kDa to 129 kDa while preserving the essential DNA-binding and cleavage functions, thereby enabling AAV packaging
2Reliability
If zinc finger nucleases are used for gene therapy, then clinical trial progress is achieved, but only single target site cleavage and low specificity are possible
Solution Approach 1:
The patent implements multiplex targeting by designing a system where a single Cas9 protein can be guided to multiple different DNA target sequences through the use of multiple distinct guide RNAs. This allows simultaneous cleavage of multiple viral DNA targets (HBV, HSV, HPV) within the same cell, providing universal applicability across different viral infections
3Ease of manufacture
If AAV vectors are used for gene delivery, then preferred gene delivery capability is achieved, but packaging capacity is restricted to approximately 4.8 kb
Solution Approach 1:
The patent changes the physical parameter of the Cas9 protein by reducing its molecular weight through truncation. This parameter change allows the Cas9 protein to fit within the AAV packaging capacity constraint while maintaining its essential nuclease functions, enabling delivery of the complete CRISPR system via AAV vectors
4Measurement precision
If ZFNs are used for gene therapy, then single locus targeting is achieved, but low specificity and inability to target multiple loci simultaneously are limitations
Solution Approach 1:
The patent segments the viral genome into multiple targetable loci by designing multiple guide RNAs that recognize different sequences within the viral DNA. This segmentation approach allows simultaneous targeting of multiple viral genes (such as HBV surface antigen, core antigen, and polymerase genes) to achieve complete viral genome disruption through cumulative cleavage events
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enhances gene editing efficiency, allows for multiplex targeting, and effectively inhibits viral replication and gene expression, offering a promising treatment for viral infections and cancers by enabling the removal of viral receptors and co-factors, particularly in tissues difficult to transduce with AAV vectors.
Implementation Method 1
Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). This system can be used to target DNA viruses for cleavage and eventual elimination of the virus from cells
Data Source
AI summary
Provided herein are recombinant constructs, vectors and expression cassettes including a first promoter which is suitably a tRNA promoter operably connected to a first polynucleotide encoding a first single guide RNA and a second promoter operably connected to a second polynucleotide encoding a Cas9 polypeptide. The first single guide RNA includes a first portion complementary to a strand of a target sequence of a DNA virus and a second portion capable of interacting with the Cas9 polypeptide. Also provided are codon optimized Staphylococcus aureus derived Cas9 polynucleotides and polypeptides with nuclear localization signals and optionally an epitope tag. Also provided are constructs for production of sgRNAs including a tRNA. Methods of inhibiting viral replication, inhibiting expression of a target sequence from a virus or treating a viral infection or viral induced cancer using the compositions are also provided.